The long-term objective of this laboratory is to determine how viruses cause disease. Coxsackievirus B4 (CB4) was chosen for these studies since this virus has been implicated in human diseases such as type 1 insulin-dependent diabetes and myocarditis. Using a mouse model system of CB4-induced pancreatitis, this proposal addresses two main issues. The first two specific aims are straightforward and focus on the molecular basis of pathogenicity: 1. Mapping of the viral gene(s) that determine the virulent phenotype using recombinant, chimeric viruses generated from cDNA clones of a non-virulent (CB4-P) and a highly virulent parental virus (CB4-V); 2. Identification of the nucleotide sequences that determine the virulent phenotype by DNA sequence analysis of appropriate cDNA clones. Since multiple mutations are likely, individual loci will be analyzed by resection of single, double, etc. mutations back into the avirulent, parental virus clone. The third specific aim begins to explore the more complex question of how specific mutations determine virulence by examining whether viral function and immunogenicity are altered. 3A. Do CB4-V and CB4-P infect different cell types within the pancreas? This question will be addressed by using in situ hybridization to detect viral RNA in infected tissue. 3B. Do the altered loci encode antigenic determinants? The appropriate peptides will be tested to determine if they can evoke a cytotoxic T cell response or an antibody response in infected mice. The fourth specific aim is proposed as an alternative to specific aim #3. 4. Characterization of the CTL response to viral infection by: A. Mapping of the CTL recognition determinants of CB4-P and CB4-V, B. Determining whether there is a difference in the CTL response against CB4-V between mice of different H-2 haplotypes, C. Determining if the tissue damage observed in CB4-V-infected mice is immune mediated. These studies should generate new data on the molecular pathogenesis of coxsackievirus infections in addition to information on the relationship between virulence and viral function and virulence and immunogenicity.